P-Glu-D-Phe-Trp-Ser-Tyr-D-Arg-Leu-Arg-Pro-Gly-NH{HD 2 {B and intermediates

ABSTRACT

D-Phe2-D-Arg6-LRF, is described as well as its synthesis by solid phase techniques and novel intermediates formed by such synthesis. The novel decapeptide exhibits anti-ovulatory activity in mammals.

United States Patent 1 Foell Dec. 30, 1975 1 P-GLU-D-PHE-TRP-SER-TYR-D-ARG-LEU- ARG-PRO-GLY-NH2 AND INTERMEDIATES [75] Inventor: Theodore J. Foell, King of Prussia,

[73] Assignee: American Home Products Corporation, New York, NY.

221 Filed: Nov. 4, 1974 21 1 Appl. No.: 520,591

[52] US. Cl. ..260/l12.5 LH; 424/177 [51] Int. Cl. C07C 103/52; A61K 37/00 [58] Field of Search 260/1 12.5

[56] References Cited I UNITED STATES PATENTS 3,855,199 l2/l974 Foell et al 260/! 12.5

Primary Examiner-Lewis Gotts Assistant Examiner-Reginald .l. Suyat [57] ABSTRACT 8 Claims, No Drawings P-GLU-D-PHE-TRP-SER-TYR-D-ARG-LEU-ARG- PRO-GLY-NH AND INTERMEDIATES called LRF) is the decapeptide', L-'(5-oxoprolyl)-L'-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-glycyl-L-leucyl-L- arginyl-L-prolylglycineamide. This decapeptide is secreted by the hypothalamus and carried to the adenohypophysis where it stimulates the release of the luteinizing hormone and the follicle stimulating hormone. In (i) copending application Ser'. No. 402,958 filed Oct. 3, 1973, now Pat. No. 3,855,199, D-Phe -D-Ala -LRF is described and claimed as having antiovulatory activity; (ii) copending application Ser. No. 417,983 filed Nov. 21, 1973, now Pat. No. 3,886,137, D-Phe -D-Leu is described and claimed as having anti-ovulatory activity and (iii) copending application Ser. No. 459,513 filed Apr. 10, 1974, now Pat. No. 3,886,l 35, D-Phe -D-Pgl LRF is described and claimed as having anti-ovulatory activity. US. Pat. No. 3,824,227 describes D-Phe -LRF as an antagonist of LRF in vitro. Other modifications of LRF are described by Fujino et al., Biochemical and Biophysical Research Communications, 49, No. 3' pp 698-705 (Nov. i972).

The present invention concerns itself with further structural modifications of LRF which exhibit antiovulatory activity. Y.

The novel peptides of the present invention are represented by the compounds of the formula:

p-(billfi-D-Phe-Trp-Ser-Tyr-D-Arg-Leu-ArgPro-Glyl 2 and its non-toxic salts. All chiral amino acid residues identified in formula I supra, and the other formulas hereinafter are of the natural or L-corifig'uration unless specified otherwise.

Also contemplated within the scope of the present invention are intermediates of the formula R-p-Glu-D- Phe-Trp-Ser(R)-Tyr(R )-D-Arg(N- R")-l.eu-Arg(N"'-R)-Pro-Gly-R wherein:

R is selected from the class consisting of NH,, OH, O- (lower)alkyl, in which (lower)alkyl is C through C (e.g. methyl, ethyl, pentyl, hexyl, etc.) and O-benzyl;

N means the side chain nitrogen atoms of arginine;

R and R are each a protecting group for the N 5 N w and N nitrogen atoms of arginine selected from the class consisting of nitro, tosyl, benzyloxycarbonyl, adamantyloxycarbonyl and tertbutyloxycarbonyl; or R is hydrogen which means thereare no protecting groups on the side chain nitrogen atoms of arginine. Where the protecting group is nitro or tosyl, the protection is on either one of the N N nitrogens and in the case of benzyloxycarbonyl, or adamantyloxycarbonyl, the protection is on the N nitrogen and either one of the N N w nitrogen atoms. The preferred protecting group definedvby, R and R is nitro;

R is at protecting group for the phenolic hydroxyl group of tyrosine selected from the class consisting of tetrahydropyranyl, tert-bu tyl, trityl, benzyl, 2,6-v dichlorobenzyl, benzyloxycarbonyl and '4- bromohenzyloxycarbonyl. The preferred protect- 2 ing-group is benzyl; or R is hydrogen which means there is no protecting group on the phenolic hy droxy function; -R" is aprotecting group for the alcoholic hydroxyl group of serine and is selected from the class coni I sisting of acetyl, benzoyl, tetrahydropyranyl, tertbutyl, trityl, benzyl, 2,6-dichlorobenzyl, or R is hydrogen which means there is no protecting group on the alcoholic oxygen atom. Preferably R is benzyl; R is preferably hydrogen or an a-amino protecting group. The a-amino protecting group contemplated by R are those known to be useful in the art in the step-wise synthesis of polypeptides. Among the classes of a-amino protecting groups covered by R are l acyl type protecting groups illustrated by the following: fortnyl, trifluoroacetyl, phthalyl, toluenesulfonyl (tosyl), benzensulfonyl, nitrophenylsulfenyl, tritylsulfenyl, o-nitrophenoxyacetyl, chloroacetyl, acetyl, 'y-chlorobutyryl, etc.; (2) aromatic urethan type protecting groups illustrated by benzyloxyearbonyl and substituted benzyloxycarbonyl such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl;. (3) aliphatic urethan protecting groups illustrated by tert-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; (4) cycloalkyl urethan type protecting groups illustrated by cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbo nyl; (5) thio urethan type protecting groups such as phenylthiocarbonyl; (6) alkyl type protecting groups as illustrated by triphenylmethyl (trityl), benzyl; (7) trialkylsilane groups such as trimethylsilane. The preferred a-amino protecting group defined by R are selected from the class consisting of tert-butyloxycarbonyl, cyclopentyloxycarbonyl, tert-amyloxycarbonyl and d-isobornyloxycarbonyl. ln formula ll at least one of R, R R and R is a protecting group. t

A further aspect of the present invention relates to intermediates linked to a solid resin support. These intermediates are represented by the formula:

R")-Leu-Arg(N-R)-Pro-Gly-A (lll) wherein:

R, R R, R and R have the same meaning as in Formula ll;

A" is an anchoring bond used in solid phase synthesis linked to a solid resin support. A" is selected from the class consisting of:

(lll

'esidue (Gly) is joined through a covalent carbon to iitrogen or oxygen bond to these phenyl rings. The alkyl chains are cross linked at approximately every iftieth carbon by p-substituted phenyl residues derived rom divinyl benzene.

In selecting a particular side chain protecting group be used in the synthesis of the peptides of formula I), the following rules should be followed: (a) the )rotecting group must be stable to the reagent and lnder the reaction conditions selected for removing the t-amino protecting group at each step of the synthesis, b) the protecting group must retain its protecting )roperties (i.e. not be split off under coupling condiions), and (c) the side chain protecting group must be emovable upon the completion of the synthesis conaining the desired amino acid sequence under reaction :onditions that will not alter the peptide chain.

Illustrative of pharmaceutically acceptable non-toxic alts of formula I are hydrochloride, hydrobromide, ulfate, phosphate, maleate, acetate, citrate, benzoate, uccinate, malate, ascorbate, and the like.

The peptides of formula (I) through (111) are preared using solid phase synthesis. The synthesis is comnenced from the C-terminal end of the peptide using 111 a-amino protected resin. Such a starting material :an be prepared by attaching an a-amino protecting ;lycine to a benzhydrilamine resin, a chloromethylated esin or a hydroxymethyl resin, the former being preerred. The preparation of a benzhydrilamine resin is lescribed by P. Rivaille et al., Helv. 54, 2772 (1971) ind the preparation of the hydroxymethyl resin is decribed by Bodanszky et al., Chem. Ind (London) 38, 597-98 (1966). A chloromethylated resin is commer- :ially available from Bio Rad Laboratories Richmond, Ialif. and the preparation of such a resin is described ry Stewart et al., Solid Phase Peptide Synthesis Freeman & Co. San Francisco 1969), Chapter I, pp -6. In using the benzhydrilamine resin an amide an- :horing bond is formed with the a-amino protected ;lycine as follows:

Till

P ystyrene resin support his permits the C-terminal amide function to be obained directly after the amino acid sequence in the ynthesis is complete by cleaving off the resin support 0 form the glycine amide at the C-terminal portion of he desired peptide of formula (I). When the other esins are used, the anchoring bond is the benzylester roup as defined supra in Formula ([1112), which after leavage of the peptide from the resin support must be onverted to the C-terminal amide. The preferred proedure is to ammonalyse the protected peptide off the esin and then remove the protecting group by hydroenolysis or by hydrogen fluoride cleavage. An alterate procedure would be to cleave by transesterificaion with methanol/(EthN and then convert the resultig ester into an amide and subsequently deprotect as escribed above. See J. M. Stewart Solid Phase Pep- .de Synthesis, pp 42-46 (W. H. Freeman & Co. 968).

The a-amino protected glycine is coupled to the enzhydrilamine resin with the aid of a carboxyl group activating compound such as dicyclohexylcarbodiimide. Following the coupling of the a-amino protected glycine to the resin support, the a-amino protecting group is removed such as by using trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone or HCl in dioxane. The deprotection is carried out at a temperature between about 0C and room temperature. Other standard cleaving reagents and conditions for removal of specific a-amino protecting groups may be used as described in Schroder & Lubke, The Peptides, 1 72-75 (Academic Press 1965). After removal of the a-amino protecting group the remaining a-amino protected amino acids are coupled step-wise in the desired order to obtain a compound of formula (1). However, as an alternate to adding each amino acid separately to the reaction, some of them may be coupled prior to addition to the solid phase reactor. If the C-terminal end of the peptide unit is represented by glycine or proline and the coupling is carried out with DCC, a minimum of racemization is encountered with proline and no problems are encountered with glycine which has no asymmetric centre. Each protected amino acid or amino acid sequence, is introduced into the solid phase reactor in about a four-fold excess and the coupling is carried out in a medium of dimethylformamide: methylene chloride (1:1) or in dimethylformamide or methylene chloride alone. In cases where incomplete coupling occurred the coupling procedure is repeated before removal of the a-amino protecting group, piror to the coupling of the next amino acid to the solid phase reactor. The success of the coupling reaction at each stage of the synthesis is monitored by the ninhydrin reaction as described by E. Kaiser et al., Analyt. Biochem, 34, 595 (1970).

After the desired amino acid sequence has been synthesized, the peptide is removed from the resin support by treatment with a reagent such as hydrogen fluoride which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups and the a-amino protecting group (if present) on pyroglutamic acid to obtain directly a compound of formula I in the case where the benzhydrilamine resin was used. Where a chloromethylated resin is used the peptide may be separated from the resin by methanolysis after which the recovered product is chromatographcd on silica gel and the collected fraction subject to ammonalysis to convert the methyl ester to the C-terminal amide. Any side chain protecting group may then be cleaved as previously described or by other procedures such as catalytic reduction (e.g. Pd on C) using condi tions which will keep the Trp moiety intact. When using hydrogen fluoride for cleaving, anisolc is included in the reaction vessel to prevent the oxidation of labile amino acid (e.g. tryptophan).

The solid phase synthesis procedure discussed supra is well known in the art and has been essentially described by M. Monahan et al., C. R. Acad. Sci. Paris; 273 508 (1971).

The nomenclature used for peptides is described by Schroder & Lubke, supra, pp viii-xxix and in Biochemistry 11, 1726-1732 (1972).

The following examples are illustrative of the preparation of the compounds of formulas I through 111.

EXAMPLE 1 L-Pyroglutamyl D-phenylalanyl-L-tryptophyl-O-benzyl-seryl-O-benzyl-L-tyrosyl-N-nitro-D arginyl-L-leucyl-N-nitro-L-arginyl L-prolylglycyl benzhydrilamine resin Benzhydrilamine resin (30.0 g.) is put in a lvlerrifieldv vessel of 300 ml. capacity and put through the following wash cycle: (a) methylene chloride; (b) trifluoroacetic acid (3 times for minutes each); (c) methylene chloride; (d) methanol; (e) triethylamine 12.5% in dimethylformamide (2 times for 10 minutes each); (f) methanol (two times); (g) methylene chloride (two times), allowing a contact time of at least 3 minutes each, if not indicated otherwise. i

The resin so prepared is then gently shaken with t-butyloxycarbonyl glycine (5.475 g., 31.2 mmole) in 1:1 methylene chloride-dimethylformamide and 38.4 ml of 1M dicyclohexylcarbodiimide in methylene chloride is added in three portions over a period of 30 minutes. Shaking is continued at ambient temperature for a total of 18 hours. The peptide-resin is then washed successively with methanol, methylene chloride, methanol (twice), and methylene chloride (twice). To test for completeness of reaction, the peptide resin is subjected to a ninhydrin test following the procedure of E. Kaiser et al., Analytical Biochemistry 34, 595 1970 The deprotection of the attached amino acid is carried out as follows: The peptide-resin is treated with a 1:1 solution of trifluoroacetic acid-methylene chloride (three times for minutes each), thensteps (c) through (g), as described above for the wash cycle are performed. Again a sample of the peptide-resin is subjected to a ninhydrin test to check for completeness of reaction. Thesample is now strongly positive indicating deprotection of the glycine molecule attached to the resin.

The following amino acid residues are then introduced consecutively: t-Boc-L-proline (31.2 mmoles, 38.4 DCC), t-Boc-N-nitro-L-arginine--(31.2 mmoles, 38..4 mmoles DCC), t-Boc-L-leucine (31.2 mmoles, 38.4 mmoles DCC).

.Each coupling step is carried out in a medium of methylene chloride dimeth ylformamide( 1:1) and the removal of the a-amino protecting group at each step is performed as described for the deprotection of the t-Boc-glycine-resin. However, after the addition of t-Boc-tryptophan the deprotection reaction is carried out with the addition of 5% 1,2-ethanedithiol added to the trifluoroacetic acid-methylene chloride medium. At this point, the washed tetrapeptide-resin is dried, weighed (33.73 g.), and the synthesis continued with 6.0 g. of the tetrapeptide-resin. The next four amino acids added are t-Boc-N-nitro-D-arginine (6.2 mmoles, 8 mmoles DCC), t-Boc-O-benzyl-L-tyrosine (6.2 mmoles, 8 mmoles DCC), t-Boc-O-benzyl-L- serine (6.2 mmoles, 8 mmoles DCC) and t-Boc-L-tryptophan (6.2 mmoles, 8 mmoles DCC). At this point, the washed octapeptide-resin is dried, weighed (8.00 g. and the synthesis continued with one third (2.66 g.) of the octapeptide-resin.

The next amino acid added is t-Boc-D-phenylalanine (2.08 mmoles, 2.7 mmoles DCC), then L-2-pyrrolidone-S-carboxylic acid. (3.12 mmoles, 4.0 mmoles DCC). The washed peptide-resin is dried in vacuo.

EXAMPLE 2 L-Pyroglutamyl-D-phenylalanyl-L tryptophy1-L-seryl- L-tyrosyl D-arginyl L-leucyl-L-arginyl-L-prolylglycinamide EXAMPLE 3 Purification and characterization of L-Pyroglutamyl-Dphenylalanyl-L-tryptophyl-L-seryl- L-tyrosyl-D-arginyl-L-1eucyl-L-arginyl-prolylglycinamide acetate The crude peptide from Example 2 is dissolved in a minimum volume of 0.2N acetic acid, applied to a Bio-Gel P-2 200-400 mesh gel filtration column (2.5 cm X cm) and eluted with the same solvent. Fractions, of 9 ml. each are collected. The fractions containing the desired peptide are located by Pauly spot test and UV analysis. After pooling and lyophilization, a white fluffy powder (526 mg.) is obtained.

A partition column of Sephadex G-25 fine (2.5 X 90 cm) is prepared by equilibration with lower phase and then upper phase of the BAW solvent system (nbutanolzacetic acid:water 4:1:5, V =l65 ml.).

The lyophilized peptide from above is applied in a minimum volume of upper phase. Elution with upper phase (6 ml. fractions) affords the desired product which is located as described above. After pooling and lyophilization, a white fluffy powder (68.7 mg.) is obtained.

A second BAW column yielded 44.4 mg. of desired product. I

The optical rotation is measured on a Carl Zeiss LEP A-2 photoelectric precision polarimeter, [01],, =24.36 (c=0.9975, 1% acetic acid); Amino acid analysis. gives the following ratios: Ser (1.06), Glu (1.02), Pro (0.77), Gly (1.12), Leu 1.00), Tyr (0.84), Phe (0.87), Trp (0.72), Arg (1.97).

The Rf value of the peptide (20 pg load) in two TLC systems (silica plates-Brinkman) visualized by ultraviolet light, iodine vapor, and Pauly reagent: n-butanol:acetic acid:water (4:1:5, upper phase), Rf 0.28; nbutanohethyl acetatezacetate acid:water (l:l:l:l), Rf 0.65.

The compounds of formula I possess anti-ovulatory activity and hence are potentially useful in inhibiting fertility in female mammals. in tests conducted with female rats (225 to 250 grams body weight) complete ovulation inhibition was achieved in 60% of the rats tested at a dose of about 24 mg/kg. The test was conducted with mature Sprague-Dawley rats, normally cycling, unanesthetized, proestrous rats. On the afternoon of proestrous, each rat in the test group received six subcutaneous injections of the acetate salt .of formula l in corn oil, each injection being given a half hour following the previous injection. The rats aresacrificed the next morning and the number of animals ovulating and the number of ova shed are recorded following the 7 procedure described by E. S. France, Neuroendocrinology 6, pp 77-89 (I970). The absence of or a significant decrease in the number of ova is the criterion for an anti-ovulation effect. At a dose of 1 mg per injection inhibition of ovulation was achieved in 60% of the rats tested.

The compounds of formula I can be administered to mammals intravenously, subcutaneously, intramuscularly or orally for fertility inhibition and control. The effective dosage will vary with the form of administration and the particular species of mammal to be treated. A typical dosage is a physiological saline solution containing a compound of formula I administered in a dose range of between about 20 to 30 mg/kg of body weight. Oral administration may be in either solid or liquid form.

What is claimed is:

l. A compound selected from the group consisting of L-p-Glu-DPhe-L-Trp-L-Ser-L-Tyr-D-Arg-L-Leu-L- Arg-L-Pro-Gly-NH and and its non-toxic salts; wherein R is selected from the class consisting of NH OH,

O (lower)alkyl and O-benzyl; R and R are selected form the class consisting of hydrogen and a protecting group for the N 5 N and N nitrogen atoms of arginine selected from nitro, tosyl, benzyloxycarbonyl and adamantyloxycarbonyl;

R is selected form the class consisting of hydrogen and at protecting group for the phenolic hydroxyl group of tyrosine selected from tert-butyl, tetrahydropyranyl, trityl, benzyl, 2,6-dichlorobenzyl, benzyloxycarbonyl and 4-bromobenzyloxycarbonyl;

R is selected from the class consisting of hydrogen and a protecting group for the alcoholic hydroxyl group of serine and is selected from acetyl, benzoyl, tetrahydropyranyl, tert-butyl, trityl, 2,6- dichlorobenzyl, benzyl and benzyloxycarbonyl;

R is selected from the class consisting of hydrogen and an a-amino protecting group, with the proviso that at least one of R, R, R and R is a protecting group.

2. A compound according to claim 1 wherein R is 3. A compound according to claim 1 wherein R is NH R and R are nitro, R is 2,6-dichlorobenzyl, R is benzyl and R is hydrogen.

4. A compound according to claim 1 which is selected from: L-Pyroglutamyl-D-phenylalanyl-L-tryptophyl-L-seryl-L-tyrosyl-D-arginyl-L-leucyl-L-arginyl- L-prolylglycinamide and its non-toxic acid addition salts.

wherein:

R and R are selected from the class consisting of hydrogen and a protecting group for the N N and N nitrogen atoms of arginine selected from nitro, tosyl, benzyloxycarbonyl and adamantyloxycarbonyl;

R is selected from the class consisting of hydrogen and a protecting group for the phenolic hydroxyl group of tyrosine selected from tert-butyl, tetrahydropyranyl, trityl, benzyl, 2,6-dichlorobenzyl, benzyloxycarbonyl and 4-bromobenzyloxycarbonyl;

R is selected from the class consisting of hydrogen and a protecting group for the alcoholic hydroxyl group of serine and is selected from acetyl, benzoyl, tetrahydropyranyl, tert-butyl, trityl, 2,6- dichlorobenzyl, benzyl and benzyloxycarbonyl;

R is selected from the class consisting of hydrogen and an a-amino protecting group; and A is selected from the class consisting of R are nitro, R is 2,6-dichlorobenzyl and R is benzyl. =l 

1. A COMPOUND SELECTED FROM THE GROUP COMSISTING OF L-P-GLU-D-PHE-L-TRP-L-SER-L-TYR-D-ARG-L-LEU-L-ARG-LPRO-GLY-NH2 AND R4-P-GLU-D-PHE-L-TRP-L-SER(R3)-L-TYR(R2)-D-ARG(NGR5)-L-LEU-L-ARG(NG-R1)-L -PRO-GLY-R AND ITS NON-TOXIC SALTS; WHEREIN R IS SELECTED FROM THE CLASS CONSISTING OF NH2, OH, O(LOWER)ALKYL AND O-BENZYL; R1 AND R5 ARE SELECTED FROM THE CLASS CONSISTING HYDROGEN AND A PROTECTING GROUP FOR THE N ,N AND N NITROGEN ATOMS OF ARGININE SELECTED FROM NITRO, TOSYL, BENZYLOXYCARBONYL AND ADAMANTYLOXYCARBONYL; R2 IS SELECTED FORM THE CLASS CONSISTING OF HYDROGEN AND A PROTECTING GROUP FOR THE PHENOLIC HYDROXYL GROUP OF TYROSINE SELECTED FROM TERT-BUTYL, TETRAHYDROPYRANYL, TRITYL, BENZYL, 2,6-DICHLOROBENZYL, BENZYLOXYCARBONYL AND 4BROMOBENZYLOXYCARBONYL; R3 IS SELECTED FROM THE CLASS CONSISTING OF HYDROGEN AND A PROTECTING GROUP FOR THE ALCOHOLIC HYDROXYL GROUP OF SERINE AND IS SELECTED FROM ACETYL, BENZOYL, TETRAHYDROPYRANYL, TERT-BUTYL, TRITYL, 2,6-DICHLOROBENZYL, BENZYL AND BENZYLOXYCARBONYL; R4 IS SELECTED FROM THE CLASS CONSISTING OF HYDROGEN AND AN A-AMINO PROTECTING GROUP, WITH THE PROVISO THAT AT LEAST ONE OF R1, R2, R3 AND R5 IS A PROTECTING GROUP.
 2. A compound according to claim 1 wherein R is NH2.
 3. A compound according to claim 1 whereIn R is NH2, R1 and R5 are nitro, R2 is 2,6-dichlorobenzyl, R3 is benzyl and R4 is hydrogen.
 4. A compound according to claim 1 which is selected from: L-Pyroglutamyl-D-phenylalanyl-L-tryptophyl-L-seryl-L-tyrosyl-D-arginyl-L -leucyl-L-arginyl-L-prolylglycinamide and its non-toxic acid addition salts.
 5. A compound of the formula: R4-L-p-Glu-D-Phe-L-Trp-L-Ser(R3)-L-Tyr(R2)-D-Arg(NG-R5)-L-Leu-L-Arg(NG-R1)-L-Pro-Gly-A wherein: R1 and R5 are selected from the class consisting of hydrogen and a protecting group for the N , N and N nitrogen atoms of arginine selected from nitro, tosyl, benzyloxycarbonyl and adamantyloxycarbonyl; R2 is selected from the class consisting of hydrogen and a protecting group for the phenolic hydroxyl group of tyrosine selected from tert-butyl, tetrahydropyranyl, trityl, benzyl, 2, 6-dichlorobenzyl, benzyloxycarbonyl and 4-bromobenzyloxycarbonyl; R3 is selected from the class consisting of hydrogen and a protecting group for the alcoholic hydroxyl group of serine and is selected from acetyl, benzoyl, tetrahydropyranyl, tert-butyl, trityl, 2,6-dichlorobenzyl, benzyl and benzyloxycarbonyl; R4 is selected from the class consisting of hydrogen and an Alpha -amino protecting group; and A is selected from the class consisting of
 6. A compound according to claim 5 wherein R4 is an Alpha -amino protecting group which is selected from the class consisting of tert-butyloxycarbonyl, cyclopentyloxycarbonyl, tert-amyloxycarbonyl and isobornyloxycarbonyl.
 7. A compound according to claim 5 wherein A is a benzhydrilamine resin and R4 is hydrogen.
 8. A compound according to claim 7 wherein R1 and R5 are nitro, R2 is 2,6-dichlorobenzyl and R3 is benzyl. 